Mapping Functional Interactions in a Heterodimeric Phospholipid Pump

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dc.contributor.authorPuts, Catheleyne F.
dc.contributor.authorPanatala, Radhakrishnan
dc.contributor.authorHennrich, Hanka
dc.contributor.authorTsareva, Alina
dc.contributor.authorWilliamson, Patrick
dc.contributor.authorHolthuis, Joost C. M.
dc.date.accessioned2021-12-23T16:14:31Z-
dc.date.available2021-12-23T16:14:31Z-
dc.date.issued2012
dc.identifier.issn00219258
dc.identifier.urihttps://osnascholar.ub.uni-osnabrueck.de/handle/unios/11115-
dc.description.abstractType 4 P-type ATPases (P-4-ATPases) catalyze phospholipid transport to generate phospholipid asymmetry across membranes of late secretory and endocytic compartments, but their kinship to cation-transporting P-type transporters raised doubts about whether P-4-ATPases alone are sufficient to mediate flippase activity. P-4-ATPases form heteromeric complexes with Cdc50 proteins. Studies of the enzymatic properties of purified P-4-ATPase.Cdc50 complexes showed that catalytic activity depends on direct and specific interactions between Cdc50 subunit and transporter, whereas in vivo interaction assays suggested that the binding affinity for each other fluctuates during the transport reaction cycle. The structural determinants that govern this dynamic association remain to be established. Using domain swapping, site-directed, and random mutagenesis approaches, we here show that residues throughout the subunit contribute to forming the heterodimer. Moreover, we find that a precise conformation of the large ectodomain of Cdc50 proteins is crucial for the specificity and functionality to transporter/subunit interactions. We also identified two highly conserved disulfide bridges in the Cdc50 ectodomain. Functional analysis of cysteine mutants that disrupt these disulfide bridges revealed an inverse relationship between subunit binding and P-4-ATPase-catalyzed phospholipid transport. Collectively, our data indicate that a dynamic association between subunit and transporter is crucial for the transport reaction cycle of the heterodimer.
dc.description.sponsorshipEuropean UnionEuropean Commission; Dutch Organization of Sciences (NWO-CW)Netherlands Organization for Scientific Research (NWO); Utrecht High Potential Program; National Science FoundationNational Science Foundation (NSF); This work was supported by grants from the European Union Framework V Program (the Flippase Project), the Dutch Organization of Sciences (NWO-CW), the Utrecht High Potential Program (to J. C. M. H.), and the National Science Foundation (to P. W.).
dc.language.isoen
dc.publisherAMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
dc.relation.ispartofJOURNAL OF BIOLOGICAL CHEMISTRY
dc.subjectAMINOPHOSPHOLIPID TRANSPORT
dc.subjectASYMMETRY
dc.subjectBiochemistry & Molecular Biology
dc.subjectCDC50 PROTEINS
dc.subjectCRYSTAL-STRUCTURE
dc.subjectP-TYPE ATPASES
dc.subjectPLASMA-MEMBRANE
dc.subjectROLES
dc.subjectSUBCELLULAR-LOCALIZATION
dc.subjectTRANSLOCATION
dc.subjectYEAST
dc.titleMapping Functional Interactions in a Heterodimeric Phospholipid Pump
dc.typejournal article
dc.identifier.doi10.1074/jbc.M112.371088
dc.identifier.isiISI:000308579800045
dc.description.volume287
dc.description.issue36
dc.description.startpage30529
dc.description.endpage30540
dc.contributor.orcid0000-0003-3663-8236
dc.contributor.orcid0000-0001-8912-1586
dc.contributor.researcheridAAH-7284-2021
dc.contributor.researcheridAAL-9157-2020
dc.identifier.eissn1083351X
dc.publisher.place11200 ROCKVILLE PIKE, SUITE 302, ROCKVILLE, MD, UNITED STATES
dcterms.isPartOf.abbreviationJ. Biol. Chem.
dcterms.oaStatusGreen Published, hybrid
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